Download Table 2

Fig-AnnotForm : On this annotation form appear, from top left to bottom right, an ISH image, information and
links about the gene (Ralgps2), the colored synthetic view showing the colored expression values of each tissue.
Then the formulars allowing to approve the annotation and to add new images. Finally , for the 25 tissues listed
on the right (tissue 6 and more are not shown here) , the radio button to select the expression level within
Negative, Weak, Medium and Strong, the select box to choose one or several keywords, and a button to add a
free comment.
Fig-Sieve : This html formular allows to create a “sieve”. The user is invited to select expression values for
several tissues (listed on the right). A click on the Search button will list all genes for which there is no
expression in the Middle ear (lines 2, 3, 4) and medium or strong expression in the Kolliger (line 9) and weak or
medium or strong expression in the Sensory retina (line 22).
The And and Or check buttons allow to choose between And or Or combination between the tissues (lines).
There is always a Or combination for the expression levels (columns) (i.e. medium Or strong)
This sieve can be saved for future use (button Save).
Fig-Dendogram: Tissue dendrogram, based upon a hierarchical clustering using average linkage of the
differentially expressed genes. Three main branches can be distinguished:



The Red branch is related to genes from tissues mainly derived from sensory regions of the inner ear.
The Green branch is more heterogeneous, although genes are mainly derived from mesenchyme tissues
such as different kinds of bones from the ribs and the otic capsule and secretary organs such as the stria
vascularis. This branch also includes the endolymphatic organ from the inner ear and the choroïde
plexus from the hindbrain as well as mesenchyme tissues from the inner ear and middle ear.
The blue branch is mainly composed of genes present in nervous tissues such as the retina, and the
hindbrain and from ectodermal/mesenchymal derived tissues from the middle ear and the follicles of
vibrissae.
The heatmaps show the expression of the genes present in all the tissue of the corresponding branch. The
columns of the heatmaps appear in the same order as the leaves of the tree in clockwise order from “T8 Inner
Spiral sulcus” up to “T21 Hindbrain”. Each line of a heatmap shows the expression in all 25 tissues. The black
vertical lines delimit the 3 main branches ‘red’, green’ and ‘blue’. To get a sufficient resolution for the display
only 1/8 lines are displayed for red and blue branches, 1/4 for the green one.
A REFAIRE AVEC LES BONNES COULEURS
Fig-PercentageCorrelation: Percentages of expressed genes from 2000 genes analyzed by in situ hybridization.
Percentages are given relative to the level of labeling in the sensory region of five sensory organs: the retina, the
olfactory organ, the vibrissae follicles, the Kölliker’s organ in the basal cochlear canal, and the utricular macula
from the vestibular organ
Fig-DiaVenn: Figure B gives a quick view of the 25 possible combinations to compare two sensory organs
(K vs K, K vs U, …, V vs R, …). To get more expressive view the 623 genes (KUROV) present in all five
organs are not taken into account. Each Venn’s diagram shows two cercles which intersect. The area of the
circles as well as the area of the intersection are proportional to the number of genes present in the organs (dark
grey and light grey) and to the number of genes present in both organs (white). For instance for U K, 253
corresponds to the number of genes presents in the Utricule (253=876-623), 162 to the genes present in the
Kölliker’s organ (162=785-623) and 80 to their common genes (80=703-623).
These diagram show that the Utricule and the Kolliker’s organ have fewer genes in common with the retina. At
the opposite the retina, the olfactory organ and the vibrisse follicule share a larger number of genes in common.
Fig-ISH: Examples of gene expression patterns in the sensory organs.





A & B: Two expressed genes in the basal cochlear canal. The cochlear canal is delineated by dashed
lines: Ko: Kölliker’s organ presents in the ventral region, Iss: inner spiral sulcus may includes the
prospective Reissner’s membrane and the outer spiral sulcus (Oss), Oc: otic capsule, sagital section.
The patchy expression of Ctgf (Connective tissue growth factor) seems to be restricted to the Kölliker’s
organ extending toward the outer spiral sulcus (Oss) and the otic capsule (Oc). Interesting enough in the
Kölliker‘s organ a region without expression (arrow) separates what could be the greater epithelial ridge
from the lesser epithelia ridge. The transcript expression of Shc3 (Src homology 2 domain-containing
transforming protein C3) is visible in the basal canal of the cochlea and restricted to a small region of
the Kölliker’s organ (arrows). Scale bar : 50 µm.
C & D: Expression of two genes in the utricule from the vestibular part of the inner ear. The expression
of the Cd9 (Cd 9 antigen) is visible in the prospective sensory region (Sr) of the utricule as well as the
non-sensory region (Nsr), (large arrow). The two horizontal arrows points toward the separation
between the sensory region and the non-sensory region. Sagital sections. Scale bar: 100 µm.
E & F: Two examples of transcripts expressed in the retina:Mid1 (Midline 1) and Fubp1 (Far upstream
element (FUSE) binding protein 1). Rpe: the retinal pigmented epithelium. Scale bar: 0.5 mm.
G & H: Two samples of expressed gene as observed on sagital sections of the olfactory organs. The
gene Gp38 (Glycoprotein 38 or podoplanin) is observed in the olfactory epithelium (Oe) and the
cartilage primordia of turbinate bones (Ct). The probe for Igsf4a (Immunoglobulin superfamily,
member 4A, transcript variant 2) is present all over the olfactory organ including the respiratory
epithelium (Re). Scale bar: 200 µm.
I: Sagital sections on the primordia of vibrissae follicles. Depending of the level of the section in the
upper lip, different regions of the vibrissae present a mRNA expression. The Mif gene (Macrophage
migration inhibitory factor) is expressed in several regions of the vibrissae follicles including the
follicle cells. Expression is also observed outside the vibrissae in the surrounding mesenchyme (Me).
Scale bar: 0.5 mm.
Fig-GO2000andKUROV: Comparison between the distribution of gene function by using the GO database
between the initial pool of 2000 genes and those from KUROV.
A: Several important functions appear in the pool of 2000 genes, the five most represented being Protein
binding, Ion binding and three other groups with approximately the same percentage: Nucleic acid binding,
Nucleotide binding, and Hydrolase activity. Out of the 2000 genes 359 were not found in the GO database, while
503 were not analyzed included in the Others & no GO section. The distribution of GO annotations for the 623
genes of KUROV show a larger percentage of Proteins binding, as well as the next three groups of proteins.
Looking in more detail at the distribution of Protein binding annotations, the percentage of Enzymes is more
important for KUROV, as well as for Transcription factor binding proteins, Proteins C-terminus, Heat shock,
Unfolded proteins and Glycoproteins, while the percentage of Receptors is reduced.
B: Comparison between the main protein activities from the initial 2000 genes (blue bars) and those from
KUROV (red bars). This analysis shows that proteins involved in channel activities such as Ion channel activity
and G protein-coupled receptor activity are in lower number in the KUROV pool of genes.
Fig-GOOlfactory: Comparison of the distribution of gene functions using GO annotation between genes
expressed or not expressed in the olfactory organ. To make possible the comparison between the two sets of gene
products, one expressed in the olfactory epithelium (1175 transcripts) and the other scored negative (825
transcripts), the number of genes was equalized to a same reference number: 1000. Gene products from two
cellular functions were compared, one related to basic cellular function involved during development such as the
Ligase activity and Transcription cofactor activity, and one in relation with receptor activity such as G-protein
coupled receptor activity and Ion channel activity. Gene products involved in basic cellular functions during
development such as Cytoskeletal protein bindings were more represented in the pool of genes expressed in the
olfactory organ. At the opposite, gene products involved in receptor activity were less numerous in this pool of
genes.
Fig-NetworkKUROV : Network analysis of genes expressed in the five sensory organs. Genes that were found to
be expressed in the five sensory organs were grouped in the pool termed “KUROV”, where K stands for the
Kölliker's organ, U for the sensory region of the utricle, R for the sensory retina, O for the olfactory organ and V
for the vibrissae follicles. For this analysis we have used the STRING database along with Cytoscape in order to
find the possible interactions between the 623 genes that are common to the five sensory tissues.

A: Out of 623 genes, only 163 presented direct interactions where they could be grouped into 7 major
networks, although other less important networks were also found. The first network is mainly
composed of ribosomal proteins and mitochondrial ribosomal proteins showing numerous interactions.
This network is linked through Eif2s1 (Translation initiation factor2, subunit 1alpha) to a second
network, whose genes are involved in protein processing. The first network is also connected to a third
one trough Rps27l (Ribosomal protein S27 like), where genes are linked to several cellular processes
such as focal adhesion, cytoskeleton regulation and neural development. This network is related to a
fourth one through Aurka (Aurora kinase A) where several proteosome genes are present.
Csnk1a1involved in Wnt signaling, and Hedgehog signaling pathways makes the connection with the
fifth network mainly made of genes involved with tubulin, actin, centrosomal and cilia proteins. A sixth


network independent from the previous ones is made of genes mainly involved in transcription. Another
(seventh) independent network, includes several transcription factors.
B: Examples of mining trough the STRING database for two genes from the fifth and the seventh
networks: Cep57and Lmo4. The Cep57 gene encodes a centrosomal protein. This gene is connected
directly with 5 genes from KUROV, three of them belonging to the tubulin family, and is indirectly
connected to a pool of 92 target genes.
C: Lmo4 (LIM domain only 4) is directly related to 7 genes present in KUROV, and via a single
connection to 148 outside target genes.
Fig-Crebbp : Expression of Crebbp (a gene involved in Rubinstein-Taybi syndrome) in the five sensory organs
and its relationship with other genes. A-E: ISH expression patterns in the basal cochlear canal (A) and the utricle
(B), both delineated by dashed lines, the retina (C), the olfactory organ (D) and the upper lip (E) where several
vibrissae follicles are present. F: STRING analysis showing direct interactions of Crebbp with 11 genes present
in KUROV. G: Crebbp and the eleven KUROV genes directly connected to it show interactions with two initial
genes with 169 other genes from the STRING database. H: diagram showing the single relation of Crebbp and
the 11 genes from KUROV with 187 genes as found from the STRING database.
Fig-NetworkOlfactory : Network analysis of genes outside KUROV, expressed in the olfactory sensory region.
For this analysis we have used the STRING database in order to find possible interactions between 552 genes
expressed in the olfactory organ (but not belonging to the KUROV list), Cytoscape for displaying the results
along with the KEGG database for possible functional pathways. Out of 552 genes only 61presented direct
interactions where the majority of them can be grouped into 2 major networks and 6 minor ones. The first major
network (1) can be divided into two sub-networks, with genes involved in Jak-STAT signaling, insulin signaling
and inositol phosphate signaling pathways. The other sub-network includes genes involved in axon guidance,
focal adhesion or actin cytoskeleton and neurotrophin function. The second network (2) can also be divided into
two sub-networks involved in centrosomal proteins, in cellular processes and in Wnt and Tgfpathways. The second sub-network is mainly involved in cell cytoskeleton. The 3rd network (3) is composed of
genes involved in nuclear pore complex, centromere and kinetochore, while the 4 th (4) is involved in the
spliceosome and in nuclear transport. The 5th network (5) is mainly composed of calcium channel voltage
dependent and of synaptic vesicle gene like. The 6th network (6) is composed of transcription factors, while the
last ones (7, 8) are mainly made of genes involved in the MAPK signaling pathway, or encoding ribosomal
proteins.
Tables
Table1
All tissues
Middle & Inner Ear Mesenchyme
Otic capsule
Stato-acoustic ganglion
Cochlear canal (ventral region)
Cochlear canal (dorsal region)
Vestibular sensory region
Vestibular non-sensory region
Semi-circular canals
Endolymphatic duct & sac
Choroïde plexus 4th ventricule
Hindbrain
Sensory retina
Olfactory organ
Cartilage primordium of Ribs
Vibrisse follicles
Table 1 Gene expression in different tissues
Number & percentage of genes
No signal
weak
medium
331 (16.5%)
1629 (81.4% 970 (48.5%)
791 (39.5%)
917 (45.8%) 403 (20.1%)
1277 (63.8%) 449 (20.9%) 205 (10.2%)
905 (45.2%)
739 (36.9%) 289 (14.5%)
1053 (52.6%) 613 (30.6%)
153 (7.6%)
1417 (70.8%) 786 (39.3%) 212 (10.6%)
1107 (55.3%) 669 (33.4%)
193 (9.6%)
1124 (56.2%) 676 (33.8%)
168 (8.3%)
1112 (55.6%) 650 (32.5%)
197 (9.8%)
1389 (69.4%) 470 (23.5%)
97 (4.8%)
1022 (51.1%) 651 (32.5%) 264 (13.2%)
454 (22.7%)
794 (39.7%) 570 (28.5%)
824 (41.2%)
723 (36.1%) 378 (18.9%)
818 (40.9%)
830 (41.5%) 298 (14.9%)
1216 (60.8%) 571 (28.5%)
153 (7.6%)
894 (44.7%)
685 (34.2%) 326 (16.3%)
strong
362 (18.1%)
90 (4.5%)
59 (2.9%)
75 (3.7%)
18 (0.9%)
33 (1.6%)
26 (1.3%)
25 (1.2%)
30 (1.5%)
12 (0.6%)
57 (2.8%)
182 (9.1%)
61 (3.0%)
47 (2.3%)
49 (2.4%)
80 (4.0%)
Legend Table 1
The ventral region of the cochlear canal mainly corresponds to the Kölliker’s organ and the
inner spiral sulcus.
The dorsal region of the cochlear canal includes the stria vascularis and the outer spiral
sulcus.
The vestibular sensory region includes the sensory cells and supporting cells of the utricule,
saccule and cristae, and may be ISH signals from some transitional cells.
The vestibular non-sensory region corresponds to transitional cells, dark cells and other cells
surrounding the vestibular sensory epithelium of the utricule, saccule and cristae.
The percentage in parenthesis is related to the 2000 genes including few cases where it was
impossible to decipher with confidence any signal for a particular tissues due to poor
histology.
Table 2
Type of sensory
epithelia
K: Kölliker's organ
U: Utricular macula
R: Sensory retina
O: Olfactory organ
V: Vibrisse follicles
Table 2
Number of gene not expressed or expressed in the five sensory tissues
Not expressed
Expressed
weak
medium
strong
Total
1215 (60.7%)
1114 (55.7%)
824 (41.2%)
818 (40.9%)
894 (44.9%)
613 (30.6%)
664 (33.2%)
723 (36.1%)
830 (41.5%)
685 (34.2%)
153 (7.6%)
187 (9.3%)
378 (18.9%)
298 (14.9%)
326 (16.3%)
18 (0.9%)
25 (1.2%)
61 (3.0%)
47 (2.3%)
80 (4.0%)
784 (39.2%)
876 (43.8%)
1162 (58.1%)
1175 (58.7%)
1091 (54.5%)
Specific genes
9 (0.4%)
11 (0.5%)
71 (3.5%)
74 (3.7%)
56 (2.8%)
Legend Table 2
Specific genes are those only present in a sensory epithelia.
The percentage in parenthesis is related to the 2000 genes including few cases where it was impossible to
decipher with confidence any signal for a particular tissues due to poor histology.